ME1 Regulates NADPH Homeostasis to Promote Gastric Cancer Growth

Cancer Tumor Biology and Immunology Research

ME1 Regulates NADPH Homeostasis to Promote Gastric Cancer Growth and Metastasis Yun-Xin Lu1,2, Huai-Qiang Ju1, Ze-Xian Liu1, Dong-Liang Chen1,2,YunWang1,2, Qi Zhao1, Qi-Nian Wu1,2, Zhao-lei Zeng1, Hai-Bo Qiu1,3, Pei-Shan Hu1, Zhi-Qiang Wang1,2, Dong-Sheng Zhang1,2, Feng Wang1,2, and Rui-Hua Xu1,2

Abstract

Genomic alterations of tumor suppressors often encompass collateral protein-coding Glutamine Promotes cell survival and growth in nutrient- genes that create therapeutic vulnerability deprived regions to further inhibition of their paralogs. Here, we report that malic enzyme 2 (ME2) is fre- Aspartate

quently hemizygously codeleted with SMAD4 ME2 SMAD4 Malate NADPH in gastric cancer. Its isoenzyme ME1 Deletion was upregulated to replenish the intracellular reducing equivalent NADPH and to Compensation ME1 ME1 ROS maintain redox homeostasis. Knockdown of ME1 signiﬁcantly depleted NADPH, ETV4 ETV4 ETV4 RE ME1 induced high levels of reactive oxygen spe- ETV4 RE ME1

cies (ROS), and ultimately cell apoptosis Nucleus Pyruvate NADP+ Stomach under oxidative stress conditions, such as Cytoplasm Promotes anchorage- glucose starvation and anoikis, in ME2- independent growth underexpressed cells. Moreover, ME1 pro- for metastasis ME1 maintains redox homeostasis to support cell survival under nutrient deprivation and anchorage-independent growth moted tumor growth, lung metastasis, and when its paralogue ME2 is codeleted with SMAD4. peritoneal dissemination of gastric cancer © 2018 American Association for Cancer Research in vivo. Intratumoral injection of ME1 siRNA significantly suppressed tumor growth in cell lines and patient-derived xenograft–based models. Mechanistically, ME1 was transcriptionally upregulated by ROS in an ETV4-dependent manner. Overexpression of ME1 was associated with shorter overall and disease-free survival in gastric cancer. Altogether, our results shed light on crucial roles of ME1-mediated production of NADPH in gastric cancer growth and metastasis. Significance: These findings reveal the role of malic enzyme in growth and metastasis. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/8/1972/F1.large.jpg. Cancer Res; 78(8); 1972–85. 2018 AACR.

Introduction genomes as well as in other mammals (1). However, vast targeted therapies often focused on the amplified, overex- Multifaceted sequencing has revealed an unprecedentedly pressed, or mutant-driving oncoproteins (2, 3), whereas the detailed blueprint for gene amplification or deletion in human deleted, underexpressed, or mutant-inactivated tumor suppressors received less attention (4). Recently, strategies such as 1 Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in synthetic lethality have been proposed to exploit genomic loss South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, of suppressor genes, as these events often occur at large regions China. 2Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou, China. 3Department of Gastric and Pancreatic Surgery, Sun Yat-sen that may encompass critical fundamental housekeeping genes – University Cancer Center, Guangzhou, China. that are essential for cell growth and survival (4 8). Cancer cells may sometimes tolerate these stresses by rewiring the informa- Note: Supplementary data for this article are available at Cancer Research fl Online (http://cancerres.aacrjournals.org/). tion ow into functionally redundant paralogs that maintain these essential cellular reactions (6, 8, 9). When several other Corrected online July 11, 2019. homologous genes serving overlapping functions were shut Y.-X. Lu, H.-Q. Ju, and Z.-X. Liu contributed equally to this article. down by inhibitors, the cells would experience lethal strike Corresponding Authors: Rui-Hua Xu, Sun Yat-sen University Cancer Center, 651 (4, 6, 10). Genetic and pharmacologic studies have evidenced Dongfeng Load East, Guangzhou, Guangdong 510060, China. Phone: 8620- the therapeutic exploit of collateral deletion in the tumor- 8734-3295; Fax: 8620-8734-3333; E-mail: [email protected]; and Feng Wang, suppressive loci (4, 10–12). One notable example is that [email protected] hemizygous deletion of TP53 in colorectal cancer necessarily doi: 10.1158/0008-5472.CAN-17-3155 led to high vulnerability to inhibition of the neighboring gene 2018 American Association for Cancer Research. POLR2A (5).

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Malic enzymes are responsible for oxidative decarboxylation Bioinformatic analysis of malate to pyruvate, which is the primary substrate supporting Gene copy number and corresponding gene expression the tricarboxylic acid (TCA) cycle and the major source of was analyzed using data obtained from CCLE (http://www. intracellular reducing equivalents (13, 14). In human cells, malic broadinstitute.org/ccle) and TCGA (http://www.cbioportal. enzymes are encoded by three homologous genes (15, 16), org/portal/) according to previously described methods (11). including ME1, which is located in the cytoplasm and enzymic We searched potential target gene in the proximity of SMAD4 activities require NADP; malic enzyme 2 (ME2), which is located gene and analyzed its codeletion with SMAD4 in gastric cancer as in the mitochondrion and enzymic activities require NAD; and in a recent report (6). ME3, which is located in the mitochondrion and enzymic activities require NADP. These enzymes are widely distributed in Protein extraction, immunoblotting, and antibodies nature and have highly conserved sequences and similar struc- Proteins were extracted with the RIPA lysis buffer (Cell Signal- tural topologies across different species, suggesting that they have ing Technology, cat. no. 9806). Briefly, scrapped cells were col- important biological functions (17). lected after centrifugation at 2,000 rpm for 3 minutes. Then, the For chemical work, there is an equally important role for ATP pelleted cells were lysed in RIPA buffer containing proteinase and and NADPH, which powers the redox defense and reductive phosphatase inhibitors for 15 minutes on ice. The supernatant biosynthesis (18). Tumor cells reprogram their metabolic patterns was transferred to a new tube, and the protein concentrations were to satisfy the needs of rapid cell proliferation at the expense of measured using BCA Protein Assay Kit (Thermo Fisher Scientific, overproduced reactive oxygen species (ROS), which requires cat. no. 23225). SDS-PAGE and immunoblotting was performed plenty of NADPH supplementation (19). By recycling the TCA as described previously (22). The following antibodies were intermediate malate into the common carbon source pyruvate, used: ME1 (Abcam, cat. no. ab97445); ME2 (Abcam, cat. no. malic enzymes may have a regulatory role in satisfying cellular ab139686); ME3 (Abcam, cat. no. ab172972); vinculin (Abcam, demand for reducing equivalents, energy, and biosynthetic pre- cat. no. ab129002); G6PD (Abcam, cat. no. ab993); PHGDH cursors (16). Malic enzymes are essential for NADPH production (Abcam, cat. no. ab211365); Flag (Abcam, cat. no. ab49763); from both the oxidative pentose phosphate pathway (16) and E-cadherin (Cell Signaling Technology, cat. no. 3195); N-cadherin glutamine metabolism (20) and thus have been evaluated as (Cell Signaling Technology, cat. no. 13116); cleaved PARP (Cell therapeutic targets. It has been reported that ME1 produces Signaling Technology, cat. no. 5625); cleaved caspase-3 (Cell NADPH at levels as high as those produced by G6PD in the Signaling Technology, cat. no. 9664); ETV4 (Aviva Systems pentose phosphate pathway shunt (18). Repression of ME1 or Biology, cat. no. ARP32263_P050); ETV4 (Lifespan Biosciences, ME2 results in altered metabolism (13, 14), reduced cell growth, cat. no. LS-B1527); and b-actin (Cell Signaling Technology, migration, and elevated ROS level in nasopharyngeal carcinoma cat. no. 4970). (13) and non–small cell lung cancer (21). To mine the therapeutic targets with collateral lethality in RNA extraction and qPCR analysis gastric cancer, we analyzed The Cancer Genome Atlas (TCGA) Total RNA was isolated from cells or tissues by TRIzol Reagent and Cancer Cell Line Encyclopedia (CCLE) databases and found (Invitrogen, cat. no. 15596018). One microgram of RNA for each that the housekeeping gene ME2 was frequently codeleted or sample was reversed to cDNA by a Prime Script RT Master Mix Kit counderexpressed with the tumor suppressor gene SMAD4 in (Takara, cat. no. RR036Q), and 1 mL cDNA was used as a template gastric cancer. During energy stress such as glucose deprivation, to perform qPCR with GoTaq qPCR Master Mix (Promega, cat. no. anchorage-independent growth, and solid tumor formation A6002) according to the manufacturer's instructions. Primers in vivo, ME1 played essential roles in supplying NADPH for used in our study were listed in Supplementary Table S1. elimination of intracellular ROS when its paralog ME2 was suppressed due to coalteration with SMAD4. Tissue specimens and clinicopathologic characteristics The total 207 paraffin-embedded, archived gastric samples used Materials and Methods in this study were histopathologic and clinically diagnosed at the Cell culture Sun Yat-sen University Cancer Center between 2007 and 2009. GES1, AGS, SGC7901 (originally purchased from ATCC on Written informed consent was obtained from all patients, and no July 2014) and SNU216, BGC823, HGC27, MGC803, NUGC4, patient received any chemo- or radiotherapy prior to surgery. The MKN45, MKN74 (originally purchased from the Institute of use of clinical specimens for research purposes was conducted in Basic Medical Sciences of the Chinese Academy of Medical accordance with the Declaration of Helsinki and approved by the Sciences on June 2014) were cultured in RPMI1640 or DMEM ethical committee of Sun Yat-sen University Cancer Center. The medium (Invitrogen) supplemented with 10% FBS (HyClone) clinicopathologic characteristics of the samples are summarized in Supplementary Table S2. All patients were followed up regularly at 37 Cwith5%CO2 according to the suppliers' instructions. Glucose-free RPMI1640 medium (GIBCO/Thermo Fisher Sci- after the operation at 3-month intervals. The median follow-up entific, cat. no. 11879020) supplemented with 10% dialyzed time was 49 months (range, 3–102 months). Fifty freshly collect- FBS (GIBCO/Thermo Fisher Scientific, cat. no. 26400-044) was ed gastric cancer tissues and matched adjacent nontumoral gastric used for glucose deprivation assays. All cells were tested neg- tissues from the same patient were frozen and stored in liquid ative for mycoplasma and authenticated by short tandem nitrogen until required for RNA or protein extraction. repeat DNA fingerprinting at the Medicine Lab of the Forensic Medicine Department of Sun Yat-sen University (Guangzhou, IHC and TUNEL analysis China). All cell lines have not been passaged for more than 6 IHC assays were conducted as reported previously (5). Briefly, months in our study after resuscitation. the sections were deparaffinized and rehydrated before they were

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heated at a subboiling temperature in sodium citrate buffer Reporter assay (pH 6.0) for 10 minutes with a microwave oven for antigen The dual reporter construct expressing Gaussia luciferase under retrieval. Samples were then incubated with 3% hydrogen per- the human ME1 promoter, and secreted alkaline phosphatase oxide for 10 minutes to block endogenous peroxidase activity and (SEAP) under the CMV promoter (used for transfection normal- then with antibody against ME1 (Abcam, ab97445, 1:500), ME2 ization) was from GeneCopoeia, Inc (cat. no. HPRM23418- (Abcam, cat. no. ab139686), Ki-67 (Cell Signaling Technology, PG04). The indicated cells were plated 18 hours before transfec- 9129, 1:500) or cleaved caspase-3 (Cell Signaling Technology, tion in 24-well plates and transiently transfected with 500 ng of 9664, 1:1,000) at 37C for 1 hour. The IHC Kit (Dako, cat. no. the reporter plasmid using Lipofectamine 3000. Plasmids con- K5007) including second antibody and DAB substrate was used to taining ETV4 open reading frame or siRNAs targeting ETV4 were detect protein expression according to the manufacturer's proto- transfected 24 hours later using Lipofectamine 3000. The lucif- col. Counterstaining color was carried out using hematoxylin. erase activity was determined according to the manufacturer's Assessments of the staining were scored by two experienced instructions (GeneCopoeia, Inc., cat. no. LF032) and normalized pathologists blinded to the patients' identity and clinical status. to that of the SEAP activity. In discrepant cases, a pathologist reviewed the cases and reached the consensus. Expression level was determined according to our Chromatin immunoprecipitation assay – previous report (23). The terminal deoxynucleotidyl transferase The chromatin immunoprecipitation (ChIP) assay was per- mediated nick end labeling (TUNEL) assays were performed formed with an EZ-Chip Kit (Millipore, cat. no. 17-371) following with the In Situ Cell Death Detection Kit (Roche, cat. no. the manufacturer's instruction as described previously (16). 293T 11684795910) according to the manufacturer's instructions. cells were grown to 80% confluence, and crosslinking was performed with 1% formaldehyde for 10 minutes. The cell lysates ROS, cell apoptosis detection, and measurement of NADPH were sonicated to shear DNA to sizes of 300 to 1,000 bp. Equal ROS levels were determined as described previously (24). aliquots of chromatin supernatants were incubated with anti- Briefly, cells cultured in glucose-deprived medium or in matrix 0 0 ETV4 or anti-IgG antibody (Millipore) overnight at 4 C with detachment conditions were incubated with 10 mmol/L 2 ,7 - rotation. After reverse cross-link of protein/DNA complexes to fl dichlorodihydro uorescein diacetate (H2-DCFDA, Thermo Fish- free DNA, RT-PCR was carried out using the specific primer fi er Scienti c, cat. no. D399) at 37 C for 30 minutes. Afterward, the (forward: 50-ACACCTGTCAGTTTCTACAGA-30, reverse: 50-CAT- cells were collected, washed twice in ice-cold PBS, and resus- TATTCAGAGAGAGCAGTGG-30) detecting the ETV4-binding site pended in PBS. Fluorescence was immediately measured using a on ME1 promoter region. FACScan Flow Cytometer (Beckman-Coulter). For apoptosis analysis, cells were collected and stained with Annexin V-FITC and PI In vivo (4A Biotech Co. cat. no. FXP018) before measurement with flow tumorigenesis and metastasis assays – cytometer. The intracellular levels of NADPH, total NADP, and All female BALB/c nude mice (4 5 weeks old) used in our study GSH were measured with the NADP/NADPH-Glo Kit (Promega, were purchased from the Beijing Vital River Laboratory Animal fi cat. no. G9081) or GSH/GSSG-Glo Kit (Promega, cat. no. V6612) Technology Co., Ltd. and housed in speci c pathogen-free units. according to the manufacturer's instructions. Subcutaneous mice model was performed as reported previously (22, 23). 6 m Vectors, siRNAs, cell transfection, and lentivirus production For lung metastasis model, 5 10 cells resuspended in 100 L ME1 vectors including wild-type (cat. no. EX-T8139-Lv121) of sterile PBS were injected into the tail veins of nude mice. Lung and silent mutation (cat. no. CS-T8139-Lv121-1) were purchased colonization was monitored at the indicated time point after from GeneCopoeia, Inc. The siRNAs targeting ME1 (targeting intraperitoneal injection of D-luciferin (Goldbio, cat. no. LUCK-1) sequences: GGGCATATTGCTTCAGTTC, GAGAGACAGCAATT- with a Xenogen IVIS 100 bioluminescent imaging system. Sixty fi GAACA) and ME2 (targeting sequence: CCCAGTATGGACA- days later, mice were sacri ced with cervical dislocation, and the fi CATCTTTA) were synthesized by RiboBio. The siRNAs targeting lungs were dissected out and paraf n embedded to histopatho- G6PD and PHGDH were purchased from RiboBio. All the trans- logically examine the metastatic locus. fection experiments were conducted with Lipofectamine 3000 Peritoneal dissemination ability of gastric cancer cells was (Thermo Fisher Scientific, cat. no. L3000015) as recommended. evaluated through intraperitoneal injection. In brief, SGC7901 6 SGC7901, MGC803, and HGC27 cells were transfected with (NC, sh#1, sh#2) cells (5 10 ) in 0.5 mL of PBS were injected lentiviruses containing ME1 shRNA (GenePharma, cat. no. 2016 into the peritoneal cavity of BALB/c nude mice. Mice were care- 13971), and stable cell lines were obtained after treatment with fully monitored until they were killed at 60 days after injection. 3 mg/mL puromycin for 3 to 5 days. Knockdown lentivirus Colon metastasis was examined and recorded. targeting ME2 was constructed as reported previously (16) and transfected into HGC27 cells. Patient-derived xenograft models and in vivo siRNA treatment The patient-derived xenograft (PDX)–bearing male nude mice Anoikis and soft agar colony formation assay model was raised and passaged as described previously (23, 25). Anoikis was induced by plating cells (2 106) on ultralow In brief, patient-derived tumor materials were collected in culture attachment 6-well plate (Sigma, cat. no. CLS3471-24EA). For soft medium and transferred to the animal houses on wet ice within 1 agar colony formation assay, cell suspension was mixed with hour after resection. Upon arrival, necrotic and supporting tissues 0.7% soft agar in 2DMEM containing 20% FBS in equal volume were carefully removed using sterilized surgical blades. The tumor and layered in triplicate onto 1.4% solidified agar in 2DMEM gross was cut into different fragments for several purposes, flash containing 20% FBS. After 10- to 14-day culture, colonies were frozen, paraffin embedding for histopathologic analysis. One counted under microscopy and photographed. third was flash frozen for protein extraction or stored at 80C

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for genomic profiling genomic typing and one third was fixed in and are redundant metabolic pathway due to three isoenzymes 10% neutral-buffered formalin for histopathologic examination. (Fig. 2A). Because ME2 was suppressed in gastric cancer, we The rest was implanted subcutaneously into the flank region of therefore focused on its paralogs including ME1 and ME3. West- female nude mice, and the incision was closed with surgical ern blot assays showed remarkable increase in protein levels of suture. Successfully engrafted tumor models were then passaged ME1 in the majority of gastric cancer cell lines compared with that and banked in liquid nitrogen after three passages in mice. in GES1 cells, whereas ME3 was only detected in MKN74 and For siRNA treatment analysis, PDX-bearing mice were prepared BGC823 cells (Fig. 2B). To explore collateral lethality of ME2 after subcutaneous incubation of gastric cancer tumor mass. genomic alteration in gastric cancer, lentiviruses containing short Cholesterol-modified ME1 siRNA or control siRNA (RiboBio, hairpin RNAs targeting ME1 were introduced into ME2-down- 5 nmol/kg) dissolved in diluted water were intratumorally regulated SGC7901 and MGC803 cells (Supplementary Fig. S2A). injected every 3 days for 18 days. All animal experiments were Surprisingly, intracellular NADPH as well as GSH levels were not carried out in accordance with the NIH Guide for the Care and Use affected after ME1 knockdown (Fig. 2C; Supplementary Fig. S2B). of Laboratory Animals with the approval from the Institutional We hypothesized that the resting ME2 was sufficient for NADPH Animal Care and Use Committee of Sun Yat-Sen University. production in this condition. However, cancer cells often experience nutrition stress due to insufficient vascularization (26, 27). Statistical analysis To mimic in vivo situation, we cultured cells in glucose deprivation All in vitro experiments were repeated three times or more, and medium and found that NADPH level in knockdown cells was data are presented as mean SD unless otherwise indicated. The significantly decreased compared with that in control cells (Fig. Student t test assumed two-tailed distributions to calculate sta- 2D). Glucose deprivation induced significantly elevated apopto- tistical significance between groups. Survival curves were gener- tic percentage of SGC7901 and MGC803 cells after knockdown of ated using the Kaplan–Meier method and compared using the log- ME1 (Fig. 2E), which could be restored by reintroduction of ME1 rank tests. The independent prognostic factors were identified by vector with silent mutation (Supplementary Fig. S2C and S2D) or the Cox proportional hazards regression model. ROC curve was pretreatment with NAC (Fig. 2F; Supplementary Fig. S2E). How- generated with Medcalc software. Differences were analyzed by ever, HGC27 cells with elevated ME2 expression (Fig. 1J) were GraphPad Prism 5 and P values less than 0.05 were considered to resistant to glucose deprivation, which could be completely reach statistical significance. abrogated by simultaneous silence of ME1 and ME2 (Fig. 2G; Supplementary Fig. S2F). Intracellular ROS level was remarkably elevated after knockdown of ME1 in SGC7901 and MGC803 cells Results (Fig. 2H), whereas increase of ROS (Fig. 2I) and decrease of Downregulation of ME2 in gastric cancer due to genomic NADPH level (Fig. 2J) in HGC27 cells was only observed after alterations coinhibition of ME1 and ME2. These data showed that during Genomic alteration of SMAD4 occurs frequently in human glucose deprivation, ME1 enables survival of gastric cancer cells cancers (Supplementary Fig. S1A). We screened nearby protein- with underexpressed ME2. coding genes located with SMAD4 and found that ME2 is posi- tioned approximately 100 kb upstream to SMAD4 locus (Fig. 1A). ME1 mediates anoikis resistance of gastric cancer Consistently, frequent genomic deletion of ME2 was observed in Like glucose starvation, matrix detachment elicits energy stress human cancers (Supplementary Fig. S1B). Although homozygous (9) evidenced by elevated H2O2 levels (Fig. 3A), which were deletion of SMAD4 and ME2 was found only in 4.76% of TCGA correlated to the extent of NADPH depletion (Fig. 3B). We stomach tissues, 162 of 414 (39.23%) cases bear hemizygous therefore tested whether ME1 was required for redox regulation deletion of SMAD4 and ME2 (Fig. 1A and B). Moreover, copy during anchorage-independent growth, a hallmark of cancer number and transcriptomic analyses in TCGA gastric cancer metastasis (28). Matrix detachment significantly increased intra- SMAD4 ME2 database showed positive correlations between and cellular H2O2 and decreased the NADPH level in ME1 knockdown at both the DNA and mRNA level (Fig. 1C and D). These positive SGC7901 and MGC803 cells in comparison with control cells correlations were also validated in pan-cancer cell lines from the (Fig. 3C and D). Knockdown of ME1 in SGC7901 and MGC803 CCLE database (Fig. 1E and F). To address whether genomic loss cells significantly suppressed colonies in soft agar (Fig. 3E). could lead to alteration of gene expression, we compared copy Apoptotic assays further confirmed ME1 mediated anoikis resis- numbers of SMAD4 and ME2 with corresponding mRNA level. tance (Fig. 3F). Enforced expression with silent mutated ME1 or Analysis of CCLE databases revealed that expression of SMAD4 pretreatment with NAC significantly attenuated anoikis of ME1 and ME2 was tightly correlated with their gene copy number (Fig. knockdown cells (Fig. 3F). During suspension culture, HGC27 1G and H). In a panel of gastric cancer cell lines, expression of ME2 cells with ME1 knockdown showed resistance to anoikis, and mirrored that of SMAD4 at mRNA and protein level (Fig. 1I and J). significantly elevated apoptosis was observed after concomitant Importantly, ME2 and SMAD4 were downregulated in the major- silence of ME2 (Fig. 3G–I). However, neither migration ability nor ity of gastric cancer cells, except for that in HGC27 cells, compared epithelial–mesenchymal transition markers of gastric cancer cells with that of GES1 cells (Fig. 1I and J). Our data indicated that ME2 showed differences following ME1 knockdown (Supplementary was suppressed in gastric cancer cells, probably due to hemizygous Fig. S3A and S3B). As serine metabolism and pentose phosphate codeletion with SMAD4. shunt also provided substantial NADPH in cancer cells, we next examined the possible influence of these pathways on gastric ME1 is essential for survival of gastric cancer cells under glucose cancer cell survival under glucose-deprived or anchorage-inde- deprivation pendent conditions. Knockdown of PHGDH or G6PD resulted in Malic enzymes replenish the TCA cycle and produce the major elevated apoptosis percentage in SGC7901 and MGC803 cells antioxidant NADPH via oxidation of malate to pyruvate (16, 19) under glucose-deprived or anchorage-independent conditions

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Figure 1. ME2 is suppressed in gastric cancer due to codeletion with SMAD4. A, Ideogram of chromosome 18 showing close proximity (<200 kb) of ME2 to SMAD4. Copy number alteration (CNA) of SMAD4 in gastric cancer. B, Frequencies of ME2 and SMAD4 copy number alteration in TCGA gastric cancer samples. C, Correlation of SMAD4 and ME2 copy number alteration in TCGA gastric cancer samples. D, Correlation of SMAD4 and ME2 mRNA level in TCGA gastric cancer samples. E, Correlation of SMAD4 and ME2 copy number alteration in CCLE cancer cell lines. F, Correlation of SMAD4 and ME2 mRNA level in CCLE cancer cell lines. G, Correlation of SMAD4 CNA and mRNA level in CCLE cancer cell lines as well as in gastric cancer cell lines. H, Correlation of ME2 copy number alteration and mRNA level in CCLE cancer cell lines as well as in gastric cancer cell lines. I, qPCR assays of ME2 and SMAD4 in a panel of gastric cancer cells and GES1 epithelial cells. J, Immunoblots of ME2 and SMAD4 in a panel of gastric cancer cells and GES1 epithelial cells. Pearson correlation coefﬁcient (r)andP values are displayed in B–H.

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Figure 2. ME1 is essential for cell survival during glucose deprivation. A, Overview of malic enzyme reaction. B, Immunoblots of ME1 and ME3 in a panel of gastric cancer cells and GES1 epithelial cells. Vinculin was used as a loading control. C, Measurement of NADPH/NADPþ in the indicated cells (control, sh#NC; ME1 knockdown, sh#1 or sh#2; knockdown-resistant ME1 vector, sh#1þR) cultured in normal medium. D, Measurement of NADPH/NADPþ in the indicated cells cultured in glucose deprivation medium. E, Brightfield images of SGC7901 and MGC803 cells cultured in glucose deprivation medium after knockdown of ME1. F, Cell apoptosis of indicated cells cultured in glucose deprivation medium. Representative images and quantification data are shown. G, Brightfield images and apoptotic percentage of HGC27 cells after knockdown of ME1 and ME2 cultured in glucose deprivation medium. H, Measurement of ROS level in SGC7901 and MGC803 cells cultured in glucose deprivation medium after knockdown of ME1. Measurement of ROS (I) and NADPH/NADPþ (J) in HGC27 cells after knockdown of ME1 and ME2 cultured in glucose deprivation medium. All error bars represent the SD of at least three replicates from two independent experiments. P values were determined by two-tailed t test.

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Figure 3. ME1 mediates anoikis resistance in gastric cancer. Intracellular ROS (A) or NADPH/NADPþ (B) level of indicated cells cultured in attached or detached conditions. C, Intracellular ROS level in SGC7901 and MGC803 cells cultured in detached conditions was measured after knockdown of ME1. D, NADPH/NADPþ level in the indicated cells cultured in detached conditions was measured. E, Soft agar colony formation assays in SGC7901 and MGC803 cells after knockdown of ME1. F, Representative histograms depicting apoptosis and apoptotic rate of indicated cells after 48 hours of suspension, as determined by ﬂow cytometry. G, Apoptotic rate of HGC27 cells after 72 hours of suspension. H, Immunoblots of cleaved PARP (c-PARP) in HGC27 cells after 72 hours of suspension. I, Intracellular ROS level in HGC27 cells cultured in detached conditions. All error bars represent the SD of at least three replicates from two independent experiments. P values were determined by two-tailed t test.

1978 Cancer Res; 78(8) April 15, 2018 Cancer Research

(Supplementary Fig. S3C and S3D). However, changes in apo- (Fig. 6A). We therefore asked whether glutaminolysis pathway ptosis were not as obvious as that in ME1 knockdown cells mediated by ME1 was activated by ROS overproduction. As (Supplementary Fig. S3D). These data clearly supported the shown in Fig. 6B and C, expression of ME1 was upregulated notion that ME1 could protect gastric cancer cells from anchor- under glucose deprivation medium or in matrix-detached con- age-independent growth. ditions at both the mRNA and protein level, and this effect was reversed by the antioxidant NAC, indicating a transcriptional ME1 is required for tumor growth, lung metastasis, and regulation manner of ME1 by ROS. To this end, we searched peritoneal dissemination of gastric cancer in vivo transcription factors with potential binding capacity with ME1 Knockdown of ME1 in SGC7901 cells significantly suppressed in JASPAR database and found ETV4 response elements in the tumor growth in vivo as evidenced by slowed growth curve and ME1 promoter (Fig. 6D). Interestingly, ETV4 mRNA and pro- reduced xenograft weight (Fig. 4A–C). Moreover, immunostain- tein levels mirrored that of ME1 during ROS stress (Fig. 6C; ing assays of Ki-67, TUNEL, and cleaved caspase-3 indicated that Supplementary Fig. S6A). Overexpression of ETV4 in SGC7901 tumors formed by negative control cells showed characteristic of and MGC803 cells increased ME1 protein levels (Fig. 6E), rapid proliferation and less apoptosis than that formed by ME1 whereas knockdown of ETV4 in SGC7901 and MGC803 cells knockdown cells (Fig. 4D). However, knockdown of ME1 or ME2 by siRNAs reduced both mRNA and protein levels of ME1 (Fig. alone in HGC27 cells showed no effects on tumor growth in the 6F; Supplementary Fig. S6B). Upregulation of ME1 induced by mice, whereas simultaneous ablation of ME1 and ME2 signifi- ROS was blocked by ETV4 depletion in SGC7901 and MGC803 cantly suppressed tumor growth in the subcutaneous mice model cells (Fig. 6G). Importantly, as in the case with ME1, knock- (Supplementary Fig. S4A and S4D), which was consistent with the down of ETV4 sensitized SGC7901 and MGC803 cells to in vitro results. To analyze anoikis in vivo, gastric cancer cells were glucose deprivation and detached conditions (Supplementary injected into the tail vein, and fluorescence imaging was used to Fig. S6C). These results suggest that ME1 was transcriptionally monitor lung metastasis. The majority of gastric cancer cells upregulated by ROS/ETV4 during energy stress conditions in diminished 48 hours after injection (Fig. 4E). However, gastric cancer cells. SGC7901/sh#NC cells formed remarkably large and excessive To determine whether ROS/ETV4 axis upregulated ME1 expres- lung metastatic diseases than SGC7901/sh#1 or SGC7901/sh#2 sion transcriptionally, we cloned 1.2 kb of genomic DNA cells (Fig. 4E). Hematoxylin and eosin staining of dissected lungs upstream of the transcription start site of the ME1 gene into a showed significantly more metastasis nodules in the control luciferase reporter plasmid. Redox stress significantly increased group compared with that in the knockdown groups (Fig. 4F). the luciferase activity of ME1 promoter in SGC7901 and MGC803 IHC analysis of the lung metastases confirmed effective knock- cells, which could be abrogated by NAC pretreatment (Fig. 6H). As down of ME1 with no effects on ME2 (Supplementary Fig. S5A). shown in Fig. 6I, enforced ETV4 expression induced elevated Moreover, knockdown of ME1 significantly suppressed metastasis reporter activity in SGC7901 and MGC803 cells. Knockdown of in the intestinal wall after peritoneal injection of gastric cancer ETV4 in SGC7901 and MGC803 cells reduced transcriptional cells (Fig. 4G and H). Altogether, our results indicated that ME1 is activity of ME1 reporter (Fig. 6J). ChIP-PCR analysis further critical for gastric cancer growth and metastasis. showed direct binding of ETV4 with ME1 promotor (Fig. 6K).

Intratumoral knockdown of ME1 suppresses growth of gastric Combination of ETV4 and ME1 predicts poor prognosis in cancer in vivo gastric cancer To further explore whether ME1 could be used as a therapeutic To investigate the biological role of ME1 in human gastric target in gastric cancer, we assessed the antitumor activity of ME1 cancer progression, IHC was performed to examine the protein targeting siRNA in mice bearing SGC7901 cells xenografted expression level of ME1 in 207 cases of paraffin-embedded gastric tumors and three PDXs (PDX#1–3). These PDX models were tissues (Supplementary Fig. S7A). ME1 expression was signifi- characterized with decreased ME2 and increased ME1 expression cantly increased in distant organ metastasis (M) and lymph node in the tumor tissues compared with corresponding normal tissues metastasis (Ln) tissues compared with adjacent normal tissues (Supplementary Fig. S5B). When the tumor volume reached (ANT) and paired primary tumor tissues (T; Fig. 7A and B), approximately 50 mm3, siRNAs targeting ME1 were injected supporting potential link between ME1 expression and gastric intratumorally once every other day. The growth of tumors treated cancer metastasis. Consistently, ME1 expression was markedly with siRNAs was significantly suppressed in SGC7901 cell–based overexpressed in the gastric cancer tissues compared with paired xenografts and in three PDXs compared with control group (Fig. normal gastric tissues at protein (Fig. 7A and B) and mRNA level 5A–F). The tumors developed from the ME1 siRNAs treatment (Fig. 7C). group displayed lower Ki-67 and ME1 staining, elevated TUNEL To determine the clinical relevance of ME1 in gastric cancer, signal, and cleaved caspase-3 expression than that in control archived patients were divided into high expression and low group (Fig. 5G). As PDX models closely resemble the biological expression group according to immunoscoring of ME1. Statistical characteristics and genomic landscape of human cancers at the analyses revealed that expression of ME1 was significantly corre- population level (25), our study provides clear preclinical clues lated with differentiation state (P ¼ 0.046), but not with other for developing ME1 inhibitors in gastric cancer. clinical parameters, including age, gender, tumor size, lymph node metastasis, venous invasion, perineural invasion, and ETV4 upregulates ME1 expression in gastric cancer tumor–node–metastasis (TNM) stage (Supplementary Table Tumor cells often experience oxidative stress, which could S2). In addition, Kaplan–Meier survival analysis and log-rank facilitate tumor growth by causing genomic instability and repro- test showed that ME1 overexpression was correlated with shorter gramming cancer cell metabolism (29). Glucose restriction and overall survival and disease-free survival (P < 0.001; Fig. 7D). anchorage-independent growth induced elevated H2O2 levels Univariate and multivariate analyses indicated that only

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Figure 4. Knockdown of ME1 inhibits growth and metastasis of gastric cancer in vivo. A, Tumor volume progression of xenografted subcutaneous SGC7901 (sh#NC, sh#1, sh#2) cells (n ¼ 6). Tumor growth curves were measured after injection, and tumor diameters were measured every 3 days. The values were given as mean SD. B, Photograph of dissected xenografts. C, Weight of dissected xenografts was recorded. D, Representative immunostaining of Ki-67, TUNEL, and cleaved caspase-3 (c-Cas3) in xenografted tumors. Scale bar, 50 mm. E, Representative luciferase imaging of lung metastatic cells in nude mice after knockdown of ME1. F, Representative results of hematoxylin and eosin staining (left) of metastatic lung nodules from mice injected with ME1 knockdown and control SGC7901 cells via the tail vein. Metastatic nodules under naked eyes or microscope were counted and recorded (right). G, SGC7901 cells were injected intraperitoneally and metastases in the colonic wall was recorded 60 days later. H, Dissected colons were photographed and metastatic nodules are indicated (arrows). Hematoxylin and eosin staining of colon was performed and metastatic numbers were recorded. All error bars represent the SD of results from 6 mice. P values were determined by two-tailed t test.

1980 Cancer Res; 78(8) April 15, 2018 Cancer Research

Figure 5. Intratumoral silencing of ME1 suppressed gastric cancer growth in vivo. A, Effect of intratumoral ME1 knockdown on tumor growth in mice after injection of SGC7901 cells. B, Dissected xenografts after intratumoral silence of ME1 were photographed. C, Tumor weights of xenografts were recorded. D–F, Effects of intratumoral ME1 knockdown on three PDX models. G, Hematoxylin and eosin (H&E) and immunostaining of Ki-67, ME1, TUNEL, and c-Cas3 in cell line or PDX-based xenografts. Scale bar, 50 mm. All error bars represent the SD of results from 5 mice. P values were determined by two-tailed t test.

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Figure 6. ETV4 upregulates ME1 expression in gastric cancer. A, Intracellular ROS level in SGC7901 and MGC803 cells cultured in glucose deprivation medium or in detached conditions. B, The ME1 mRNA level in SGC7901 and MGC803 cells cultured in glucose deprivation medium or in detached conditions was measured. C, Immunoblots of ETV4 and ME1 in SGC7901 and MGC803 cells cultured in glucose deprivation medium or in detached conditions. D, ETV4 DNA-binding sites are presented in the human ME1 promoter region. E, Immunoblots of ETV4 and ME1 in SGC7901 and MGC803 cells after enforced expression of ETV4. F, Immunoblots of ETV4 and ME1 in SGC7901 and MGC803 cells after siRNA-mediated knockdown of ETV4. G, Immunoblots of ETV4 and ME1 in SGC7901 and MGC803 cells cultured in glucose deprivation medium after siRNA-mediated knockdown of ETV4. H, Dual-luciferase reporter assays in SGC7901 and MGC803 cells cultured in glucose deprivation medium or in detached conditions. I, Dual-luciferase reporter assays in SGC7901 and MGC803 cells after enforced expression of ETV4. J, Dual-luciferase reporter assays in SGC7901 and MGC803 cells after knockdown of ETV4. K, ChIP-PCR in 293T cells demonstrating ME1 promoter occupancy by ETV4. Data are representative of two independent experiments. All error bars represent the SD of at least three replicates from two independent experiments. P values were determined by two-tailed t test.

1982 Cancer Res; 78(8) April 15, 2018 Cancer Research

Figure 7. ETV4/ME1 axis is overactivated in gastric cancer. A, Representative staining showed upregulated expression of ME1 protein in gastric tumor tissues compared with corresponding nontumorous tissues. Scale bar, 100 mm. B, Immunoscoring of ME1 in adjacent normal tissues (ANT), gastric cancer tissues (T), distant organ metastasis (M), and lymph node metastasis (Ln). C, The ME1 mRNA expression in 50 paired gastric tumor samples and corresponding nontumorous tissues. D, Kaplan–Meier analysis of overall survival or disease-free survival curves for gastric cancer patients with low versus high expression of ME1. E, Immunoblots of ETV4 and ME1 in eight freshly collected gastric cancer samples. F, The relative protein expression levels in eight freshly collected gastric cancer samples were quantiﬁed by comparing the gray level of each band using ImageJ Software. G, Correlations between mRNA level of ETV4 and ME1 in 50 freshly collected gastric cancer samples. H, Correlations of ETV4 and ME1 protein expression in gastric cancer tissues based on immunoscoring. I, Gastric cancer patients were divided into three groups according to immunoscoring of ETV4 and ME1, and overall survival curve was generated with Kaplan–Meier methods. J, Receiver operating characteristic (ROC) curve analysis of ME1 [area under a curve (AUC) ¼ 0.587 (95% CI, 0.517–0.655)] or ETV4 [AUC ¼ 0.672 (95% CI, 0.603–0.735)] single scoring or combinational scoring [AUC ¼ 0.785 (95% CI, 0.722–0.839)]. P values were determined by two-tailed t test. K, Proposed working model of the current study.

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upregulated ME1 expression and TNM stage were independent which was a common impediment to tumor growth (9) and prognostic factors for outcome in gastric cancer (P < 0.001; totally distinguished them from in vitro culture system. When Supplementary Table S3). its paralog ME2 was suppressed, ME1 showed a key function in We further analyzed the expression of ETV4 and ME1 in eight providing NADPH for glutathione regeneration and ROS elim- freshly collected gastric cancer samples. Western blot analysis ination, which was critical for gastric cancer cell survival under indicated that both ETV4 and ME1 were significantly upregulated energy stress conditions, such as glucose limitations, anchor- in the eight tumor samples examined, compared with the paired age-independent growth, and solid tumor formation in vivo. adjacent noncancerous tissues from the same patients (Fig. 7E). In Moreover, ME1 was transcriptionally upregulated by ROS in an addition, ETV4 expression was positively correlated with ME1 ETV4-dependent manner. Our results provided comprehensive expression at protein level (P ¼ 0.0043, r ¼ 0.6727) as analyzed in insights into the redundant roles of ME1 in gastric cancer the eight samples (Figs. 7E and F; Supplementary Fig. S7B). In 50 tumorigenesis and metastasis (Fig. 7K). freshly collected clinical gastric cancer samples, ETV4 mRNA Expression of ME1 was known to be regulated by well-known expression was statistically correlated with the mRNA levels of oncogenes or tumor suppressors such as KRAS (20, 35) or TP53 ME1 (P < 0.001, r ¼ 0.6298; Fig. 7G). Figure 7H showed that there (16). Overexpression of ME1 was reported to predict poor was a significant positive correlation between ETV4 expression prognosis of hepatocellular carcinoma (36) and to confer and ME1 (P ¼ 0.035) in the 207 gastric cancer samples. Samples radiation resistance in lung cancer (21). Suppression of ME1 that had lower level of ETV4 expression also had a lower ME1 led to glucose addiction of nasopharyngeal cancer (13) and expression, whereas samples that had higher level of ETV4 expres- colorectal cancer cells (14), which was further confirmed in our sion had a higher ME1 expression (Supplementary Fig. S7C). In study. However, Zheng FJ and colleagues found that enzymic our patient cohort, overexpression of ETV4 was significantly activity and protein level of ME1 was induced by excessive associated with outcome of gastric cancer (Supplementary Fig. carbohydrate supplementation, including glucose and pyruvate S7D). On the basis of ETV4 and ME1 expression, gastric cancer (13), whereas upregulation of ME1 was observed after glucose patients were categorized into three groups with different risks of deprivation in our study, indicating a tissue-specificregulation disease progression or death. Patients with high expression of pattern. Moreover, aberrant expression of ME1 was associated both ETV4 and ME1 showed the worst outcome (Fig. 7I; Supple- with poor prognosis in our patient cohort. Taken together, mentary Fig. S7E). Moreover, combination of ETV4 and ME1 these findings suggested that ME1 has an important function immunostaining showed higher predictive value than either in the growth and survival of cancer cells and that it could be parameter alone of gastric cancer patients survival in ROC curve used as a drug target in cancer therapy. We next explored analysis (Fig. 7J). therapeutic potential of ME1 inhibition in cell line–based as well as PDX models via in vivo siRNA treatment.SilenceofME1 significantly suppressed tumor growth and induced elevated Discussion cell apoptosis. Importantly, a recent report has revealed a panel Homozygous deletion of SMAD4 was frequently identified in of small molecules that could inhibit activities of malic nearly one third of pancreatic cancer cases (30), and loss of enzymes (17). However, further studies are needed to confirm neighboring housekeeping genes often leads to collateral lethality the clinical benefit of these inhibitors. (4, 6). However, neither homozygous deletion (31) nor inacti- In this study, we provide the genetic and pharmacologic evi- vation mutation (32) of SMAD4 was reported to be tightly dences that ME1 inhibition is lethal in cells with collateral loss of associated with gastric cancer, although knockout mice have ME2 due to hemizygous deletion of SMAD4, whereas ME2-intact clearly demonstrated its tumor-suppressive functions in the gas- cells could rely on ME2 to undergo glutaminolysis and to provide trointestinal tract (33). Our analysis identified frequent hemizy- NADPH for cell survival under redox stress conditions. Inhibition gous deletion of SMAD4 in gastric cancer and concurrent under- of ME1 would be a promising therapeutic alternative in gastric expression of ME2, which leads to high dependency of cancer cells cancer treatment. to ME1 in energy stress conditions. Cancer cells often require much more NADPH supplemen- Disclosure of Potential Conflicts of Interest tation for redox hemostasis, lipid oxidation, and biomolecular No potential conflicts of interest were disclosed. synthesis than their normal counterparts (19, 34, 35). It is predicted that intracellular NADPH comes from the oxidative pentose phosphate pathway (30%), the glutaminolysis flux Authors' Contributions through malic enzymes (30%), and the methylenetetrahy- Conception and design: H.-Q. Ju, R.-H. Xu drofolate dehydrogenase–mediated folic metabolism (40%) Development of methodology: Y.-X. Lu, H.-Q. Ju, Y. Wang, P.-S. Hu, in proliferating cells (18). We previously reported that disrup- D.-S. Zhang, F. Wang, R.-H. Xu tion of G6PD-gated pentose phosphate pathway resulted in Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.-X. Lu, H.-Q. Ju, D.-L. Chen, Y. Wang, Q.-N. Wu, marked reduction in NADPH and enhanced sensitivity to ROS H.-B. Qiu, Z.-Q. Wang, D.-S. Zhang stresses (23). In this study, we focused on malic enzymes family Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and found that ME2 was downregulated in gastric cancer due to computational analysis): Y.-X. Lu, H.-Q. Ju, Z.-X. Liu, Q. Zhao, Q.-N. Wu, concomitant genomic deletion with SMAD4 (6, 32). Cells R.-H. Xu could tolerate hemizygous deletion of ME2 as the redundant Writing, review, and/or revision of the manuscript: Y.-X. Lu, Z.-X. Liu, R.-H. Xu paralog ME1 provided metabolic anaplerosis for reducing Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.-X. Lu, Y. Wang, Q. Zhao, Z.-l. Zeng, equivalents and TCA substrates. However, metabolic stress Z.-Q. Wang, D.-S. Zhang, F. Wang, R.-H. Xu developed when tumor growth exceeded the ability of available Study supervision: F. Wang, R.-H. Xu vasculature to supply tumor cells with oxygen and nutrients, Other (assistance in generation of PDX models): Y. Wang

1984 Cancer Res; 78(8) April 15, 2018 Cancer Research

Acknowledgments The costs of publication of this article were defrayed in part by the This research was supported by National High Technology Research and payment of page charges. This article must therefore be hereby marked advertisement Development Program of China (863 Program), China (No. 2015AA020103 to in accordance with 18 U.S.C. Section 1734 solely to indicate R.-H. Xu), National Natural Science Foundation of China (nos. 81602137 to this fact. H.-Q. Ju; 81572392 to Z.-L. Zeng; 31501069 to Z.-X. Liu), Natural Science Foundation of Guangdong Province (nos. 2017A030313485 to H.-Q. Ju; 2014A030312015 to R.-H. Xu), and Science and Technology Program of Received October 14, 2017; revised December 6, 2017; accepted January 18, Guangdong (no. 2015B020232008 to R.-H. Xu). 2018; published online April 15, 2018.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Cancer Correction Research Correction: ME1 Regulates NADPH Homeostasis to Promote Gastric Cancer Growth and Metastasis Yun-Xin Lu, Huai-Qiang Ju, Ze-Xian Liu, Dong-Liang Chen, Yun Wang, Qi Zhao, Qi-Nian Wu, Zhao-lei Zeng, Hai-Bo Qiu, Pei-Shan Hu, Zhi-Qiang Wang, Dong-Sheng Zhang, Feng Wang, and Rui-Hua Xu

In the original version of this article (1), the immunostaining of Ki-67 (si#ME1 group of PDX#2 and PDX#3) in Fig. 5G was inadvertently duplicated during data arrange- ment. The image has been replaced with the intended image for Fig. 5G. The error does not affect the conclusion and has been corrected in the latest online HTML and PDF versions of the article. The authors regret this error.

Reference 1. Lu YX, Ju HQ, Liu ZX, Chen DL, Wang Y, Zhao Q, et al. ME1 regulates NADPH homeostasis to promote gastric cancer growth and metastasis. Cancer Res 2018;78:1972–85.

Published online July 15, 2019. Cancer Res 2019;79:3789 doi: 10.1158/0008-5472.CAN-19-1611 Ó2019 American Association for Cancer Research.

www.aacrjournals.org 3789 ME1 Regulates NADPH Homeostasis to Promote Gastric Cancer Growth and Metastasis

Yun-Xin Lu, Huai-Qiang Ju, Ze-Xian Liu, et al.

Cancer Res 2018;78:1972-1985.

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